MAPPING OF THE ACTIVE-SITE OF ESCHERICHIA-COLI METHIONYL-TRANSFER RNA-SYNTHETASE - IDENTIFICATION OF AMINO-ACID-RESIDUES LABELED BY PERIODATE-OXIDIZED TRANSFER RNAFMET MOLECULES HAVING MODIFIED LENGTHS AT THE 3'-ACCEPTOR END

Initiator tRNA moiecules modified at the 3′-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopie [32P]PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75. The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified. The chymotryptic digestion of the covalent MTST•[14C]tRNA-C75ox complex yielded four peptides (A-D). In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified. Peptides A, B, C. and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Vall41-Tyrl65, and Glu433-Phe437, respectively, in the MTST primary structure. In a previous work [Hountondji, C., Blanquet, S. & Lederer, F. (1985) Biochemistry 24, 1175-1180], all these peptides but one (peptide D) had been already found labeled upon MTST incubation with [14C]tRNA-A76ox. According to the crystallographic structure of MTST, the labeled residues K335, K61, K142, K147, and K149 are within a sphere of about 5.5-Å radius. The present results therefore argue for a marked flexibility of the 3′-end of the enzyme-bound tRNA, enabling it to contact any of the identified reacting residues. Such a cluster of basic amino acids may reflect ionic requirements in the guiding of the negatively charged CCA arm of tRNA toward enzyme-bound methionyl-adenylate. © 1990, American Chemical Society. All rights reserved.